The present invention relates to animal cells which can be infected by influenza viruses and are adapted to growth in suspension in serum-free medium, and to processes for the replication of influenza viruses in cell culture using these cells. The present invention further relates to the influenza viruses obtainable by the process described and to vaccines which contain viruses of this type or constituents thereof.
All influenza vaccines which have been used since the 40s until today as permitted vaccines for the treatment of humans and animals consist of one or more virus strains which have been replicated in embryonate hens"" eggs. These viruses are isolated from the allantoic fluid of infected hens"" eggs and their antigens are used as vaccine either as intact virus particles or as virus particles disintegrated by detergents and/or solventsxe2x80x94so-called cleaved vaccinexe2x80x94or as isolated, defined virus proteinsxe2x80x94so-called subunit vaccine. In all permitted vaccines, the viruses are inactivated by processes known to the person skilled in the art. The replication of live attenuated viruses, which are tested in experimental vaccines, is also carried out in embryonate hens"" eggs.
The use of embryonate hens"" eggs for vaccine production is time-, labor- and cost-intensive. The eggsxe2x80x94from healthy flocks of hens monitored by veterinariansxe2x80x94have to be incubated before infection, customarily for 12 days. Before infection, the eggs have to be selected with respect to living embryos, as only these eggs are suitable for virus replication. After infection the eggs are again incubated, customarily for 2 to 3 days. The embryos still alive at this time are killed by cold and the allantoic fluid is then obtained from the individual eggs by aspiration. By means of laborious purification processes, substances from the hen""s egg which lead to undesired side effects of the vaccine are separated from the viruses, and the viruses are concentrated. As eggs are not sterile (pathogen-free), it is additionally necessary to remove and/or to inactivate pyrogens and all pathogens which are possibly present.
Viruses of other vaccines such as, for example, rabies viruses, mumps, measles, rubella, polio viruses and FSME viruses can be replicated in cell cultures. As cell cultures originating from tested cell banks are pathogen-free and, in contrast to hens"" eggs, are a defined virus replication system which (theoretically) is available in almost unlimited amounts, they make possible economical virus replication under certain circumstances even in the case of influenza viruses. Economical vaccine production is possibly also achieved in that virus isolation and purification from a defined, sterile cell culture medium appears simpler than from the strongly protein-containing allantoic fluid. The isolation and replication of influenza viruses in eggs leads to a selection of certain phenotypes, of which the majority differ from the clinical isolate. In contrast to this is the isolation and replication of the viruses in cell culture, in which no passage-dependent selection occurs (Oxford, J. S. et al., J. Gen. Virology 72(1991),185-189; Robertson, J. S. et al., J. Gen. Virology 74 (1993) 2047-2051). For an effective vaccine, therefore, virus replication in cell culture is also to be preferred from this aspect to that in eggs.
It is known that influenza viruses can be replicated in cell cultures. Beside hens"" embryo cells and hamster cells (BHK21-F and HKCC), MDBK cells, and in particular MDCK cells have been described as suitable cells for the in-vitro replication of influenza viruses (Kilbourne, E. D., in: Influenza, pages 89-110, Plenum Medical Book Company-New York and London, 1987). A prerequisite for a successful infection is the addition of proteases to the infection medium, preferably trypsin or similar serine proteases, as these proteases extracellularly cleave the precursor protein of hemagglutinin [HA0] into active hemagglutinin [HA1 and HA2]. Only cleaved hemagglutinin leads to the adsorption of the influenza viruses on cells with subsequent virus assimilation into the cells (Tobita, K. et al., Med. Microbiol. Immunol., 162 (1975),9-14; Lazarowitz, S. G. and Choppin, P. W., Virology, 68 (1975) 440-454; Klenk, H.-D. et al., Virology 68 (1975) 426-439) and thus to a further replication cycle of the virus in the cell culture.
The Patent U.S. Pat. No. 4,500,513 described the replication of influenza viruses in cell cultures of adherently growing cells. After cell proliferation, the nutrient medium is removed and fresh nutrient medium is added to the cells with infection of the cells with influenza viruses taking place simultaneously or shortly thereafter. A given time after the infection, protease (e.g. trypsin) is added in order to obtain an optimum virus replication. The viruses are harvested, purified and processed to give inactivated or attenuated vaccine. Economical influenza virus replication as a prerequisite for vaccine production cannot be accomplished, however, using the methodology described in the patent mentioned, as the change of media, the subsequent infection as well as the addition of trypsin which is carried out later necessitates opening the individual cell culture vessels several times and is thus very labor-intensive. Furthermore, the danger increases of contamination of the cell culture by undesirable micro-organisms and viruses with each manipulation of the culture vessels. A more cost-effective alternative is cell proliferation in fermenter systems known to the person skilled in the art, the cells growing adherently on microcarriers. The serum necessary for the growth of the cells on the microcarriers (customarily fetal calf serum), however, contains trypsin inhibitors, so that even in this production method a change of medium to serum-free medium is necessary in order to achieve the cleavage of the influenza hemagglutinin by trypsin and thus an adequately high virus replication. Thus this methodology also requires opening of the culture vessels several times and thus brings with it the increased danger of contamination.
The present invention is thus based on the object of making available cells and processes which make possible simple and economical influenza virus replication in cell culture. This object is achieved by the provision of the embodiments indicated in the patent claims. The invention thus relates to animal cells which can be infected by influenza viruses and which are adapted to growth in suspension in serum-free medium. It was found that it is possible with the aid of cells of this type to replicate influenza viruses in cell culture in a simple and economical manner. By the use of the cells according to the invention, on the one hand a change of medium before infection to remove serum can be dispensed with an on the other hand the addition of protease can be carried out simultaneously to the infection. On the whole, only a single opening of the culture vessel for infection with influenza viruses is thus necessary, whereby the danger of the contamination of the cell cultures is drastically reduced. The expenditure of effort which would be associated with the change of medium, the infection and the subsequent protease addition is furthermore decreased. A further advantage is that the consumption of media is markedly decreased.
The cells according to the invention are preferably vertebrate cells, e.g. avian cells, in particular hens"" embryo cells. In a particularly preferred embodiment, the cells according to the invention are mammalian cells, e.g. from hamsters, cattle, monkeys or dogs, in particular kidney cells or cell lines derived from these. They are preferably cells which are derived from MDCK cells (ATCC CCL34 MDCK (NBL-2)), and particularly preferably cells of the cell line MDCK 33016. This cell line was deposited under the deposit number DSM ACC2219 on Jun. 7, 1995 according to the requirements of the Budapest Convention for the International Recognition of the Deposition of Micro-organisms for the Purposes of Patenting in the German Collection of Micro-organisms (DSM), in Brunswick, Federal Republic of Germany, recognized as the international deposition site. The cell line MDCK 33016 is derived from the cell line MDCK by passaging and selection with respect to the capability of growing in suspension in serum-free medium and of replicating various viruses, e.g. orthomyxoviruses, paramyxoviruses, rhabdoviruses and flavoviruses. On account of these properties, these cells are suitable for economical replication of influenza viruses in cell culture by means of a simple and cost-effective process.
The present invention therefore also relates to a process for the replication of influenza viruses in cell culture, in which cells according to the invention are used, in particular a process which comprises the following steps:
i) proliferation of the cells according to the invention described above in serum-free medium in suspension;
ii) infection of the cells with influenza viruses;
iii) addition of protease shortly before, simultaneously to or shortly after infection; and
iv) further culturing of the infected cells and isolation of the replicated influenza viruses.
The cells according to the invention can be cultured in the course of the process in various serum-free media known to the person skilled in the art (e.g. Iscove""s medium, ultra CHO medium (BioWhittaker), EX-CELL (JRH Biosciences)). Otherwise, the cells for replication can also be cultured in the customary serum-containing media (e.g. MEM or DMEM medium with 0.5% to 10%, preferably 1.5% to 5%, of fetal calf serum) or protein-free media (e.g. PF-CHO (JRH Biosciences)). Suitable culture vessels which can be employed in the course of the process according to the invention are all vessels known to the person skilled in the art, such as, for example, spinner bottles, roller bottles or fermenters.
The temperature for the proliferation of the cells before infection with influenza viruses is preferably 37xc2x0 C. Culturing for proliferation of the cells (step (i)) is carried out in a preferred embodiment of the process in a perfusion system, e.g. in a stirred vessel fermenter, using cell retention systems known to the person skilled in the art, such as, for example, centrifugation, filtration, spin filters and the like.
The cells are in this case preferably proliferated for 2 to 18 days, particularly preferably for 3 to 11 days. Exchange of the medium is carried out in the course of this, increasing from 0 to approximately 1 to 3 fermenter volumes per day. The cells are proliferated up to very high cell densities in this manner, preferably up to approximately 2xc3x97107 cells/ml. The perfusion rates during culture in the perfusion system can be regulated both via the cell count, the content of glucose, glutamine or lactate in the medium and via other parameters known to the person skilled in the art. For infection with influenza viruses, about 85% to 99%, preferably 93 to 97%, of the fermenter volume is transferred with cells to a further fermenter. The cells remaining in the first fermenter can in turn be mixed with medium and replicated further in the perfusion system. In this manner, continuous cell culture for virus replication is available.
Alternatively to the perfusion system, the cells in step (i) of the process according to the invention can preferably also be cultured in a batch process. The cells according to the invention proliferate here at 37xc2x0 C. with a generation time of 20 to 30 h up to a cell density of about 8 to 25xc3x97105 cells/ml.
In a preferred embodiment of the process according to the invention, the pH of the culture medium used in step (i) is regulated during culturing and is in the range from pH 6.6 to pH 7.8, preferably in the range from pH 6.8 to pH 7.3.
Furthermore, the pO2 value is advantageously regulated in this step of the process and is preferably between 25% and 95%, in particular between 35% and 60% (based on the air saturation). According to the invention, the infection of the cells cultured in suspension is preferably carried out when the cells in the batch process have achieved a cell density of about 8 to 25xc3x97105 cells/ml or about 5 to 20xc3x97106 cells/ml in the perfusion system.
In a further preferred embodiment, the infection of the cells with influenza viruses is carried out at an m.o.i. (multiplicity of infection) of about 0.0001 to 10, preferably of 0.002 to 0.5. The addition of the protease which brings about the cleavage of the precursor protein of hemagglutinin [HA0] and thus the adsorption of the viruses on the cells, can be carried out according to the invention shortly before, simultaneously to or shortly after the infection of the cells with influenza viruses. If the addition is carried out simultaneously to the infection, the protease can either be added directly to the cell culture to be infected or, for example, as a concentrate together with the virus inoculate. The protease is preferably a serine protease, and particularly preferably trypsin.
In a preferred embodiment, trypsin is added to the cell culture to be infected up to a final concentration of 1 to 200 xcexcg/ml, preferably 5 to 50 xcexcg/ml, and particularly preferably 5 to 30 xcexcg/ml in the culture medium. During the further culturing of the infected cells according to step (iv) of the process according to the invention, trypsin reactivation can be carried out by fresh addition of trypsin in the case of the batch process or in the case of the perfusion system by continuous addition of a trypsin solution or by intermittent addition. In the latter case, the trypsin concentration is preferably in the range from 1 xcexcg/ml to 80 xcexcg/ml.
After infection, the infected cell culture is cultured further to replicate the viruses, in particular until a maximum cytopathic effect or a maximum amount of virus antigen can be detected. Preferably, the culturing of the cells is carried out for 2 to 10 days, in particular for 3 to 7 days. The culturing can in turn preferably be carried out in the perfusion system or in the batch process.
In a further preferred embodiment, the cells are cultured at a temperature of 30xc2x0 C. to 36xc2x0 C., preferably of 32xc2x0 C. to 34xc2x0 C., after infection with influenza viruses. The culturing of the infected cells at temperatures below 37xc2x0 C., in particular in the temperature ranges indicated above, leads to the production of influenza viruses which after inactivation have an appreciably higher activity as vaccine, in comparison with influenza viruses which have been replicated at 37xc2x0 C. in cell culture.
The culturing of the cells after infection with influenza viruses (step (iv)) is in turn preferably carried out at regulated pH and pO2. The pH in this case is preferably in the range from 6.6 to 7.8, particularly preferably from 6.8 to 7.2, and the pO2 in the range from 25% to 150%, preferably from 30% to 75%, and particularly preferably in the range from 35% to 60% (based on the air saturation).
During the culturing of the cells or virus replication according to step (iv) of the process, a substitution of the cell culture medium with freshly prepared medium, medium concentrate or with defined constituents such as amino acids, vitamins, lipid fractions, phosphates etc. for optimizing the antigen yield is also possible.
After infection with influenza viruses, the cells can either be slowly diluted by further addition of medium or medium concentrate over several days or can be incubated during further perfusion with medium or medium concentrate decreasing from approximately 1 to 3 to 0 fermenter volumes/day. The perfusion rates can in this case in turn be regulated by means of the cell count, the content of glucose, glutamine, lactate or lactate dehydrogenase in the medium or other parameters known to the person skilled in the art.
A combination of the perfusion system with a fed-batch process is further possible. In a preferred embodiment of the process, the harvesting and isolation of the replicated influenza viruses is carried out 2 to 10 days, preferably 3 to 7 days, after infection. To do this, for example, the cells or cell residues are separated from the culture medium by means of methods known to the person skilled in the art, for example by separators or filters. Following this the concentration of the influenza viruses present in the culture medium is carried out by methods known to the person skilled in the art, such as, for example, gradient centrifugation, filtration, precipitation and the like.
The invention further relates to influenza viruses which are obtainable by a process according to the invention. These can be formulated by known methods to give a vaccine for administration to humans or animals. The immunogenicity or efficacy of the influenza viruses obtained as vaccine can be determined by methods known to the person skilled in the art, e.g. by means of the protection imparted in the loading experiment or as antibody titers of neutralizing antibodies. The determination of the amount of virus or antigen produced can be carried out, for example, by the determination of the amount of hemagglutinin according to methods known to the person skilled in the art. It is known, for example, that cleaved hemagglutinin binds to erythrocytes of various species, e.g. to hens"" erythrocytes. This makes possible a simple and rapid quantification of the viruses produced or of the antigen formed.
Thus the invention also relates to vaccines which contain influenza viruses obtainable from the process according to the invention. Vaccines of this type can optionally contain the additives customary for vaccines, in particular substances which increase the immune response, i.e. so-called adjuvants, e.g. hydroxide of various metals, constituents of bacterial cell walls, oils or saponins, and moreover customary pharmaceutically tolerable excipients.
The viruses can be present in the vaccines as intact virus particles, in particular as live attenuated viruses. For this purpose, virus concentrates are adjusted to the desired titer and either lyophilized or stabilized in liquid form.
In a further embodiment, the vaccines according to the invention can contain disintegrated, i.e. inactivated, or intact, but inactivated viruses. For this purpose, the infectiousness of the viruses is destroyed by means of chemical and/or physical methods (e.g. by detergents or formaldehyde). The vaccine is then adjusted to the desired amount of antigen and after possible admixture of adjuvants or after possible vaccine formulation, dispensed, for example, as liposomes, microspheres or xe2x80x9cslow releasexe2x80x9d formulations.
In a further preferred embodiment, the vaccines according to the invention can finally be present as subunit vaccine, i.e. they can contain defined, isolated virus constituents, preferably isolated proteins of the influenza virus. These constituents can be isolated from the influenza viruses by methods known to the person skilled in the art.
Furthermore, the influenza viruses obtained by the process according to the invention can be used for diagnostic purposes. Thus the present invention also relates to diagnostic compositions which contain influenza viruses according to the invention or constituents of such viruses, if appropriate in combination with additives customary in this field and suitable detection agents. The examples illustrate the invention.